Cultivation of microorganisms in culture medium, also known as growth medium, has been and continues to remain a fundamental cornerstone of the practice of microbiology, and extends back to the origins of the science. Cultivation of microorganisms is growth of microorganisms for the purposes of propagation, isolation, purification, differentiation, selection, identification, and determination of antibiotic susceptibility. It has long been recognized that microorganisms require sources of nutrients for growth. Such nutrients are provided in sterile preparations formulated to provide a nutrient environment advantageous to cultivating a microorganism of interest. Often nutrients are provided in either liquid form, commonly known as broth, or in solid form, which is usually a broth with a gelling agent such as agar, gelatin, or pectin included as an ingredient, often referred to simply as agar.
Many culture medium compositions have been developed for specific purposes and to meet specific nutritional requirements of various microorganisms, many of which exhibit specific nutritional requirements in addition to nutrients found in traditional culture media. Successful cultivation of specific microorganisms often requires supplements including blood, blood products, vitamins, and amino acid to be included in the growth medium. Cultivation processes may also require inclusion of inhibitors such as antibiotics for suppression of growth of microorganisms not of interest while permitting growth of microorganisms of interest.
Culture medium compositions have also been developed to selectively cultivate a microorganism of interest from a mixed population of microorganisms. Selective cultivation of microorganisms from a mixed population of microorganisms is most commonly accomplished by including in the culture medium one or more compounds which are inhibitory to those microorganisms in a mixture which are not of interest, yet is not inhibitory to the microorganism of interest. Antibiotics are often used as selective inhibitory compounds included in selective culture medium.
Other culture medium compositions have also been developed which differentiate a microorganism of interest from those not of interest. Differential culture medium often contains components to which various microorganisms respond differently, most often producing a visual means for distinguishing between microorganisms. For example, chromogenic substrates have been used to distinguish between microorganisms producing a specific enzyme and other which do not produce the specific enzyme, thus differentiating one from the other.
Culture medium may include a wide variety of components, for example, nutrients, essential nutrients, essential elements, growth promoters, selective inhibitors, environmental conditioners, environmental controllers, indicators of biochemical reactions, end product detectors, biochemical reaction substrates, and enzyme substrates. Culture medium recipes may be developed to improve cultivation of specific microorganisms by including components advantageous to cultivation of the specific organism of interest. Some culture medium components, while required for a specific function are prone to degradation under various environmental conditions related to preparation, transportation, or storage of a culture medium. Such environmental conditions may include, but are not limited to, temperature extremes, light exposure, atmosphere condition, pH, oxidation-reduction state, ionic state, mineral salt concentration, humidity, or moisture. Components may be particularly unstable while in a liquid environment which is commonly the physical state of culture medium.
A common problem encountered in clinical microbiology laboratories is cultivation, isolation, and identification of many pathogenic microorganisms, requires specialized culture media, which often contains components that are not stable under common storage conditions. Microorganisms causing human and animal disease, may occur infrequently and may also require specialized culture medium for propagation, selection, differentiation, identification and susceptibility testing, yet a clinical microbiology laboratory must maintain the capacity to cultivate these microorganisms on short notice. Consequently, many laboratories are faced with the inefficiency of maintaining specialized culture medium which may have a short shelf life, and which becomes degraded before it is used.
Many culture medium components are degraded or destroyed by commonly employed methods of culture medium production, which may require preparing a solution of components in a liquid diluent, most often water, sterilizing the mixture by autoclaving, steam pressurized to 15 psi, 121° C., for at least 15 minutes, followed by cooling. To avoid such component degradation, a mixture of heat labile components may be prepared and sterilized by non destructive means, for example, by filtration through a membrane filter, and the filter sterilized heat labile mixture may be added to the cooled autoclaved mixture. Some components may require no sterilization process, for example, blood, blood cells, serum, or plasma, but, nonetheless may be labile. Such components may be added directly to the autoclaved medium.
A number of commercial culture medium manufacturers provide supplement preparations for addition to culture medium in quantities sufficient to manufacture large batches of culture medium. Culture medium is routinely manufactured by preparing the culture medium in a large batch, and after cooling the culture medium, any labile supplements are added. Only after the culture medium is completed in batch, is the culture medium dispensed into individual containers such as tubes, bottles, or Petri plates used for distribution to users. These containers of culture medium are routinely used for cultivation of a single sample, specimen or microorganism and constitute a culture medium unit dose.
The process has several disadvantages. First, the manufacturing process is complex requiring careful handling, transferring, mixing and dispensing sterile products while avoiding contamination resulting in expensive, inefficient manufacturing, high potential for contamination, and difficulty in quality control. Second, this process does not allow a culture medium unit dose to be dispensed until after all components have been added to the mixture, the mixture then cannot readily or economically be resterilized. This requires the mixture be dispensed into culture medium unit doses under aseptic conditions, resulting in a high risk of contamination. Third, once a labile component has been added to the mixture, the culture medium stability or shelf life is substantially reduced. Fourth, due to a short shelf life, the end user laboratory is subject to loss of the culture medium due to degradation of the culture medium before it can be used.
Yet another manufacturing process has been to prepare culture medium as described, and then after dispensing culture medium unit doses, to dry the culture medium, requiring rehydration prior to the time of use. Culture media are dried by one of several alternative processes including forced air, vacuum, freeze drying, heat, or combinations of these processes. Drying has several disadvantages. First, it is an expensive and time consuming manufacturing process requiring extensive handling of the medium. The drying process does not lend itself to continuous processing, but requires batch processing. Additional handling steps increases the risk of contamination. Second, many culture medium components do not readily dry completely. They form a viscous liquid containing tightly bound water resulting in poor stability and difficulty in rehydrating the culture medium. Even when completely dried the components are extremely hygroscopic and easily become moist, losing stability. Third, once dried, the components may individually or collectively be resistant to dissolution in a diluent. Such components may require special procedures to dissolve in diluent, for example first dissolving in acid or an organic solvent prior to diluting with diluent. Fourth, a separate sterile diluent must be prepared and used, thereby increasing cost, complexity of use, and increased risk of contamination.
What is needed is a culture medium unit dose which offsets the problems discussed. A culture medium unit dose having its components packaged and maintained in separate component unit doses would provide the means to manufacture, package, ship, and store each composition unit dose under conditions best suited for the specific composition unit dose, for example one composition unit dose may be most stable when in a dry state, another may be most stable at freezing temperature, yet another may be most stable when protected from light, and yet another may be sufficiently stable to tolerate storage at room temperature. Further, what is also needed is a means to provide culture medium unit dose which can be maintained for long periods of time in expectation of infrequent need to use the culture medium unit dose. What is needed is a culture medium unit dose which is not constructed until the time the culture medium is needed for use. Having the culture medium unit dose packaged as individual composition unit doses, each stored for optimum stability, to be combined forming the culture medium dose at or near the time of intended use is needed. The present invention addresses these needs.